HMGB1 upregulates NF-kB by inhibiting IKB-α and associates with diabetic retinopathy

doi:10.1016/j.lfs.2019.117146

Life Sciences

Volume 241, 15 January 2020, 117146

https://doi.org/10.1016/j.lfs.2019.117146 Get rights and content

Abstract

Aims

Diabetic retinopathy (DR) is the main cause of blindness in adults and investigating new therapeutic targets for DR is necessary. This study aimed to investigate the effect of high-mobility group box 1 (HMGB1) protein and its mechanism in diabetic retinopathy (DR) were investigated.

Main methods

Human retinal endothelial cells (HREC) were uesd for chip-seq. Sprague Dawley (SD) rats were randomly divided into control group, HMGB1 group, diabetes mellitus (DM) combined with HMGB1 siRNA group, and DM group. Next, eyeballs were removed and retinas were detached for western blot. The DM model of cell was built by increasing the glucose concentration in cell culture medium. The regulation of HMGB1 was achieved by short hairpin (sh)-HMGB1 transfection, then, the transfected cells were harvested for luciferase assay, western blot and qRT-PCR analyses as well as proliferation and apoptosis detection.

Key findings

Chip-seq and luciferase assay showed the possible transcription factor functions of HMGB1 and IKB-α was one of the HMGB1 binding sites. In vivo and in vitro results indicated high expression of HMGB1 and NF-kB and low expression of IKB-α in DR and the expression of IKB-α and NF-kB was regulated by HMGB1. Moreover, cell assays showed that HMGB1 inhibited cell proliferation and promoted apoptosis.

Significance

The results from the present study showed that HMGB1 may be involved in the pathogenesis of DR as a transcription factor through NF-kB pathway. Therefore, blockade of HMGB1 may be a new method for the treatment of DR.

Introduction

Diabetic retinopathy (DR), a major microvascular complication of diabetes mellitus (DM), is the main cause of blindness in adults [[1], [2], [3]]. Because the extremely complicated pathogenesis of DR has not been fully elucidated, investigating and finding new therapeutic targets for DR is necessary. The main pathological changes of DR include retinal cell apoptosis and angiogenesis [4]. Previous studies have shown that progression of DR is accompanied by oxidative stress [5], mitochondrial dysfunction [6], and inflammation [7] as well as other clinical manifestations.

High-mobility group box 1 (HMGB1) protein is a highly conserved non-histone DNA-binding protein that plays an important role in many biological behaviors. As a late inflammatory factor, HMGB1 can be released into extracellular fluid actively or passively after stress [8,9]. In the cytoplasm, HMGB1 is involved in the imbalance of autophagy and apoptosis [10]. When released, HMGB1 can regulate angiogenesis [11], cell metastasis, and release of inflammatory cytokines [[12], [13], [14]].

Recent studies [15,16] have shown that HMGB1 is associated with diabetic peripheral neuropathy and involved in the occurrence and progression of DR; however, the underlying mechanism remains unclear. Therefore, in the present study, the specific effects of HMGB1 and its mechanism in DR were investigated.

Section snippets

Animals and groups

Forty specific-pathogen-free (SPF) male Sprague Dawley (SD) rats weighing 240–280 g were selected and randomly divided into the normal or DM group. All animal experiments were approved by the Ethics Committee of the Affiliated Hospital of China Medical University (ethics number: 2016PS229K). Rats in the DM group were injected intraperitoneally with 1% streptozototocin (STZ, Sigma, USA) at the dose of 60 mg/kg, and the rats in the normal group were injected intraperitoneally with an equal amount

Animal model

Weight and blood glucose were not significantly different among the control, HMGB1, DM combined with HMGB1 siRNA, and DM groups (p > 0.05). After 72 h of injection with STZ, the blood glucose in rats in both the DM combined with HMGB1 siRNA and DM groups increased to >16.7 mmol/L, indicating the establishment of the DM model was 100% successful. By comparison, the weight of rats in the DM combined with HMGB1 siRNA and DM groups was lower than in the control and HMGB1 groups (all p < 0.05);

Proliferation and apoptosis

After the overexpression of HMGB1, CCK-8 was used to investigate the impact of HMGB1 on proliferation of the HREC line. Cell proliferation in the sh-HMGB1 transfection group was lower than in the sh-NC transfection group (Fig. 4B). Flow cytometry results showed the apoptosis rate in the sh-HMGB1 transfection group was higher than in the sh-NC transfection group (Fig. 4A).

Discussion

HMGB1, a multifunctional protein, is associated with a variety of physiological and pathological processes, including cell proliferation and differentiation, inflammation, immunity, cancer, metabolism, and oxidative stress [[17], [18], [19], [20], [21]]. However, information regarding the function of HMGB1 in DR is relatively limited. In the present study, the HMGB1 expression in the retinas of diabetic rats at 17 weeks of age was higher than in the control group. After HREC were treated for

Conclusions

In conclusion, the results from this study showed that HMGB1 may affected the NF-kB pathway through IKB-α, thus affecting the pathogenesis of DR, and the inhibition of HMGB1 may provide a novel concept for the treatment of DR.

The following are the supplementary data related to this article.

. HMGB1 binding sites

Funding

This work was partly supported by the National Natural Science Foundation of China (81570866).

Ethics approval and consent to participate

Animal experimental procedures were in accordance with institutional guidelines and approved by the Ethics Committee of the Affiliated Hospital of China Medical University (ethics number: 2016PS229K).

Declaration of competing interest

The authors declare that there are no conflicts of interest.

References (37)

A. Gedebjerg
Prevalence of micro- and macrovascular diabetes complications at time of type 2 diabetes diagnosis and associated clinical characteristics: a cross-sectional baseline study of 6958 patients in the Danish DD2 cohort
J. Diabetes Complicat.
(2018)
S.Y. Wang et al.
Incidence and risk factors for developing diabetic retinopathy among youths with type 1 or type 2 diabetes throughout the United States
Ophthalmology
(2017)
J. Tang et al.
Inflammation in diabetic retinopathy
Prog. Retin. Eye Res.
(2011)
J.R. van Beijnum
Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature
Blood
(2006)
W. Huang et al.
HMGB1, a potent proinflammatory cytokine in sepsis
Cytokine
(2010)
X. Yang
HMGB1: a novel protein that induced platelets active and aggregation via toll-like receptor-4, NF-kappaB and cGMP dependent mechanisms
Diagn. Pathol.
(2015)
E.R. Vandermark
Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization
Virology
(2012)
A. Agarwal et al.
Diabetic retinopathy: variations in patient therapeutic outcomes and pharmacogenomics
Pharmacogenomics and Personalized Medicine
(2014)
R.A. Kowluru et al.
Role of oxidative stress in epigenetic modification of MMP-9 promoter in the development of diabetic retinopathy
Graefes Arch. Clin. Exp. Ophthalmol.
(2017)
M. Mishra et al.
Peripheral blood mitochondrial DNA damage as a potential noninvasive biomarker of diabetic retinopathy
Invest. Ophthalmol. Vis. Sci.
(2016)

P.F. Torres et al.

The role of cytokines in corneal immunopathology

Ocul. Immunol. Inflamm.

(2001)

R.C. Chen

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

Mol. Cell. Biochem.

(2014)

W.K. Kim

Identification of specifically activated angiogenic molecules in HMGB-1-induced angiogenesis

BMB Rep.

(2017)

X. Zhu

Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

J. Clin. Invest.

(2015)

G.P. Sims et al.

HMGB1 and RAGE in inflammation and cancer

Annu. Rev. Immunol.

(2010)

U. Andersson et al.

HMGB1 is a therapeutic target for sterile inflammation and infection

Annu. Rev. Immunol.

(2011)

J.O. Thomas et al.

H1 and HMGB1: modulators of chromatin structure

Biochem. Soc. Trans.

(2012)

H. Zhao et al.

HMGB-1 as a potential target for the treatment of diabetic retinopathy

Med. Sci. Monit.

(2015)

Cited by (74)

mm9_circ_014683 regulates microglia polarization through canonical NFκB signaling pathway in diabetic retinopathy
2024, Cellular Signalling
Diabetic retinopathy (DR) is still the major cause of visual loss in working-aged people, one of the critical pathological processes are retinal microglia-mediated inflammation. Our previous study demonstrated that enhanced M1 microglial polarization was involved in retinal inflammation in DR, but the detailed mechanism needs further investigation. Circular RNAs (circRNAs) are important kind of noncoding RNAs involved in the regulation of various cell biological processes. Herein, the circRNA expression profiles of BV2 mouse microglia treated with or without glucose were detected, and a total of 347 differentially expressed circRNAs were identified in glucose-treated BV2 cells. The key circRNA mm9_circ_014683 increased after glucose stimulation. Inhibiting or overexpressing mm9_circ_014683 showed no effect on the proliferation and apoptosis of microglia. Inhibiting mm9_circ_014683 impeded M1 polarization and promoted M2 polarization, and overexpressing mm9_circ_014683 showed the opposite effect. A total of 216 differentially expressed genes were identified in mm9_circ_014683-knockdown BV2 cells, which were enriched in several signaling pathways, including the NFκB signaling pathway. Moreover, mm9_circ_014683 positively regulated the canonical, NFκB signaling pathway. Besides, mm9_circ_014683 was highly expressed in the retinal microglia of diabetic mice, and intraocular injection of Lv-circRNA inhibited M1 but enhanced M2 retinal microglial polarization. In conclusion, mm9_circ_014683 regulates microglial polarization through the canonical NFκB signaling pathway in diabetic retinopathy. This study may provide insight into the pathogenesis and treatment of DR.
Effect of astrocyte GPER on the optic nerve inflammatory response following optic nerve injury in mice
2024, Heliyon
Activated astrocytes are a primary source of inflammatory factors following traumatic optic neuropathy (TON). Accumulation of inflammatory factors in this context leads to increased axonal damage and loss of retinal ganglion cells (RGCs). Therefore, in the present study, we explored the role of the astrocyte G protein-coupled estrogen receptor (GPER) in regulating inflammatory factors following optic nerve crush (ONC), and analyzed its potential regulatory mechanisms. Overall, our results showed that GPER was abundantly expressed in the optic nerve, and co-localized with glial fibrillary acidic proteins (GFAP). Exogenous administration of G-1 led to a significant reduction in astrocyte activation and expression of inflammation-related factors (including IL-1β, TNF-α, NFκB, and p-NFκB). Additionally, it dramatically increased the survival of RGCs. In contrast, astrocytes were activated to a greater extent by exogenous G15 administration; however, RGCs survival was significantly reduced. In vitro, GPER activation significantly reduced astrocyte activation and the release of inflammation-related factors. In conclusion, activation of astrocyte GPER significantly reduced ONC inflammation levels, and should be explored as a potential target pathway for protecting the optic nerve and RGCs after TON.
Research progress of the effective active ingredients of Astragalus mongholicus in the treatment of diabetic peripheral neuropathy
2024, Biomedicine and Pharmacotherapy
Diabetic peripheral neuropathy (DPN) is one of the most prevalent consequences of diabetes, with a high incidence and disability rate. The DPN’s pathogenesis is extremely complex and yet to be fully understood. Persistent high glucose metabolism, nerve growth factor deficiency, microvascular disease, oxidative stress, peripheral nerve cell apoptosis, immune factors, and other factors have been implicated in the pathogenesis of DPN. Astragalus mongholicus is a commonly used plant used to treat DPN in clinical settings. Its rich chemical components mainly include Astragalus polysaccharide, Astragalus saponins, Astragalus flavones, etc., which play a vital role in the treatment of DPN. This review aimed to summarize the pathogenesis of DPN and the studies on the mechanism of the effective components of Astragalus mongholicus in treating DPN. This is of great significance for the effective use of Chinese herbal medicine and the promotion of its status and influence on the world.
RelB mediated by GSK3β/IκBα as a potential therapeutic target in pilocarpine seizure model rats and drug-resistant epilepsy patients
2024, Journal of Neurorestoratology
Drug-resistant epilepsy (DRE) affects more than 20 million people worldwide. DRE patients do not respond to anti-seizure medications. Shifting from anti-seizure to anti-epileptic and disease-modifying therapy will be an important aim for future research. The canonical nuclear factor kappa B (NF-κB) signaling pathway plays a pivotal role in epilepsy and neuroinflammation. However, the expression and regulation of RelB, which can be activated via both canonical and non-canonical NF-κB signaling, are obscure in epilepsy. To clarify the expression and localization of RelB in the DRE phenotype and to determine the proteins related to RelB regulation, we conducted the following studies.
Quantitative PCR was performed to detect the transcription of RELB in pilocarpine-induced epileptic rats. Western blotting was used to determine the abundance of RelB and proteins related to RelB regulation in brain tissue from both epilepsy model rats and DRE patients and in liposaccharide-induced HT22 cells. Immunofluorescence staining and immunohistochemistry were used to locate RelB in brain sections from DRE patients. ELISA was used to determine inflammatory cytokines secreted by HT22 cells into the culture medium.
RELB transcription and expression were increased in the hippocampus and cortex of epileptic rats during the acute and latent phases and in epileptic foci of patients with temporal lobe epilepsy. Additionally, RelB levels were mainly increased in epileptic rat neurons and accumulated in the nuclei of hippocampal neurons. Further research demonstrated increased GSK3β phosphorylation at the Ser9 inhibitory site and decreased IκBα levels, which contributed to RelB accumulation in hippocampal neurons after seizure and in liposaccharide-stimulated HT22 cells.
This study is the first to demonstrate that RelB is widely distributed and increased in epileptogenic foci neurons in epileptic phenotypes. These results indicate that RelB may play roles in DRE and is mediated by GSK3β and IκBα, providing a new target for anti-epileptic and disease-modifying therapy.
Panax notoginseng saponins alleviate diabetic retinopathy by inhibiting retinal inflammation: Association with the NF-κB signaling pathway
2024, Journal of Ethnopharmacology
Diabetic retinopathy (DR) is a neurovascular disease that causes blindness in adults and is the most serious and common complication of diabetes mellitus. Retinal inflammation is an early stage of DR, and it is believed to play a crucial role in the development of DR. Panax notoginseng saponins (PNS) are the major active constituent in the main root of P. notoginseng, and they exhibit various biological activities, including anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory functions. However, the protective effects and underlying mechanisms of PNS against DR remain unclear.
This study aimed to investigate the alleviation effects of PNS on DR and the mechanisms involved. Furthermore, it intended to explore the major components that exert efficacy in vivo.
Streptozotocin (STZ) was administered intraperitoneally to Sprague Dawley rats, and PNS was administered orally for 1 month after 2 months of STZ injection. The morphological structure of the retina and retinal acellular capillaries were assessed via hematoxylin and eosin (H&E) staining assay. The disruption of the blood–retinal barrier (BRB) was detected through Evans blue dye leakage assay, and retinal leukocyte adhesion was achieved via fluorescein isothiocyanate-coupled concanavalin A lectin labeling assay. Immunofluorescence staining and Western blot assays were conducted to detect the expression of tight junction proteins, adhesion molecules, and the ionized calcium-binding adapter molecule-1 (Iba-1) in the retina. Enzyme-linked immunosorbent assay was performed to detect the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in serum. In addition, the protein expression levels of nuclear factor (NF)-κB p65, phosphorylated IκB kinase (p-IKK), phosphorylated NF-κB inhibitor (p-IκB), and phosphorylated NF-κB p65 (p-p65) were measured using Western blot assay. The ocular tissue distribution of PNS in normal and diabetic rats was determined through ultra-performance liquid chromatography–tandem mass spectrometry. The in vitro anti-inflammatory effects of PNS, notoginsenoside (NGR1), ginsenoside Rg1, Re, Rb1, and Rd (GRg1, GRe, GRb1, and GRd) were evaluated on human Müller (MIO-M1) cells.
PNS increased the reduction in retinal inner nuclear layer thickness, reduced the increase in retinal acellular capillaries, and attenuated elevated BRB disruption by upregulating the decrease in protein expression of claudin-1 and occludin. Furthermore, PNS significantly abrogated microglial cell activation and reversed the increase in leukocyte adhesion by downregulating the increase in the protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Moreover, PNS reduced the elevated levels of TNF-α, IL-6, and IL-1β in serum and inhibited the increased protein expression of p-IKK, p-IκB, and p-p65, and the nuclear translocation of p65. The tissue distribution results revealed that NGR1, GRg1, GRe, GRb1, and GRd were detected in the ocular tissue, while GRg1 and GRb1 were found at the highest levels compared with the other components. The cellular results showed that PNS, NGR1, GRg1, GRe, GRb1, and GRd suppressed the development of cellular inflammatory responses by inhibiting the activation of the NF-κB signaling pathway in MIO-M1 cells and that their anti-inflammatory effects were comparable.
PNS suppressed retinal inflammation by inhibiting the activation of the NF-κB signaling pathway, alleviating DR. GRg1 and GRb1 may be the primary components that exert anti-inflammatory effects in vivo.
HMGB1 in the interplay between autophagy and apoptosis in cancer
2024, Cancer Letters
Lysosome-mediated autophagy and caspase-dependent apoptosis are dynamic processes that maintain cellular homeostasis, ensuring cell health and functionality. The intricate interplay and reciprocal regulation between autophagy and apoptosis are implicated in various human diseases, including cancer. High-mobility group box 1 (HMGB1), a nonhistone chromosomal protein, plays a pivotal role in coordinating autophagy and apoptosis levels during tumor initiation, progression, and therapy. The regulation of autophagy machinery and the apoptosis pathway by HMGB1 is influenced by various factors, including the protein's subcellular localization, oxidative state, and interactions with binding partners. In this narrative review, we provide a comprehensive overview of the structure and function of HMGB1, with a specific focus on the interplay between autophagic degradation and apoptotic death in tumorigenesis and cancer therapy. Gaining a comprehensive understanding of the significance of HMGB1 as a biomarker and its potential as a therapeutic target in tumor diseases is crucial for advancing our knowledge of cell survival and cell death.

View all citing articles on Scopus

View full text

HMGB1 upregulates NF-kB by inhibiting IKB-α and associates with diabetic retinopathy

Abstract

Aims

Main methods

Key findings

Significance

Introduction

Section snippets

Animals and groups

Animal model

Proliferation and apoptosis

Discussion

Conclusions

Funding

Ethics approval and consent to participate

Declaration of competing interest

J. Diabetes Complicat.

Ophthalmology

Prog. Retin. Eye Res.

Blood

Cytokine

Diagn. Pathol.

Virology

Diabetic retinopathy: variations in patient therapeutic outcomes and pharmacogenomics

Pharmacogenomics and Personalized Medicine

Role of oxidative stress in epigenetic modification of MMP-9 promoter in the development of diabetic retinopathy

Graefes Arch. Clin. Exp. Ophthalmol.

Peripheral blood mitochondrial DNA damage as a potential noninvasive biomarker of diabetic retinopathy

Invest. Ophthalmol. Vis. Sci.

The role of cytokines in corneal immunopathology

Ocul. Immunol. Inflamm.

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

Mol. Cell. Biochem.

Identification of specifically activated angiogenic molecules in HMGB-1-induced angiogenesis

BMB Rep.

Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

J. Clin. Invest.

HMGB1 and RAGE in inflammation and cancer

Annu. Rev. Immunol.

HMGB1 is a therapeutic target for sterile inflammation and infection

Annu. Rev. Immunol.

H1 and HMGB1: modulators of chromatin structure

Biochem. Soc. Trans.

HMGB-1 as a potential target for the treatment of diabetic retinopathy

Med. Sci. Monit.